Adenovirus vectors are considered the most efficient and widely used vehicles for gene delivery. An ongoing challenge in the field of gene therapy and vaccine research is to generate liquid adenovirus formulations that are able to stabilize these viruses for longer periods of time within a realistic storage temperature range for pharmaceutical products, such as from about 2° C. to about 8° C.
Biological activity of an adenovirus depends upon the conformational integrity of at least a core sequence of nucleotides surrounded by an icosahedral capsid structure consisting of the capsid proteins. Unlike traditional organic and inorganic drugs, these are highly complex biological structures and minor chemical or physical stressors can contribute to the degradation of the adenoviral particle. A good formulation of adenovirus preparations is, therefore, of crucial importance to ensure a reasonable shelf-life, but stabilizing these vectors poses particular challenges. Adenoviruses may lose potency as a result of physical instabilities, including denaturation, aggregation (both soluble and insoluble aggregate formation), precipitation and adsorption, as well as chemical instabilities, including hydrolysis, deamidation, and oxidation. Any of these degradation routes can lead to lowered biological activity, and can also potentially result in the formation of by-products or derivatives having increased toxicity and/or altered immunogenicity.
Therefore, a tailored approach is needed to find a robust formulation for adenoviruses ensuring stability over a wide range of conditions. Buffer type, pH and specialized excipients will need to be meticulously optimized to keep an adenovirus chemically, physically and biologically stable. In view of all the factors that can be varied, finding optimal conditions for formulating adenoviruses is burdened with challenges, and the composition of a good formulation is a priori unpredictable.
Lyophilized formulations exist and are stable. However, they tend to be relatively expensive, require time-consuming handling before administration, and potency might, to a certain extent, be lost in the lyophilization process. Liquid formulations that are stable under frozen conditions (−80° C.) exist, but these require specialized shipment and expensive storage facilities, making a reliable cold chain almost impossible, especially at the periphery of the distribution network. A preferred formulation for adenoviruses is, therefore, a liquid formulation that offers adenoviral stability at a temperature range between 2° C. and 8° C., or higher. Such a formulation can be stored in a regular refrigerator and can be administered quickly and easily.
Liquid formulations for adenoviruses have been described previously, for instance, in Evans et al. 2004. The exemplified best formulations in the application are Tris-buffered formulations having a pH ranging between 7.5 and 8.5. It was found herein that the formulations are suboptimal for adenoviruses. Formulations for adenoviruses are also disclosed in WO 00/29024, which mainly relates to lyophilizing techniques. Other formulations for adenoviruses comprising a polyol are mentioned in WO 00/29024.
Accordingly, there is a need in the art to find formulations that improve the adenoviral stability by preserving quantity and potency of the contained adenovirus during storage over a prolonged period of time. The adenoviral stability should also be retained in the case of agitation stress during transport or shear forces during production or clinical use, and under wide-ranging climatic conditions, in particular, at elevated temperature or after repeated freeze/thaw cycles. Furthermore, the formulation should be suitable for the intended route of administration, should be well tolerated and should preferably have a composition with as little components as possible.